The development of new therapeutic drugs against the coronavirus causing Severe Acute Respiratory Syndrome (SARS) is a continuing imperative. Following its emergence in Guangdong Province, China, in November 2002, SARS-coronavirus (SARS-CoV) spread to other Asian countries, North America and Europe. By July 2003 there were more than 8,000 cases in 26 countries on five continents, with 774 deaths and enormous economic damage. The identification of the horseshoe bat, that is widespread throughout southern China, as the probable animal reservoir for SARS-COV indicates that there is a continuing potential for retransmission to humans and re-emergence. The initial SARS-COV epidemic was contained entirely through public health measures;there is still no effective therapeutic for SARS-COV. The replicase polyprotein is processed by two viral proteases, papain-like protease (PLpro) and chymotrypsin-like protease (3CLpro). In current work, we have demonstrated that PLpro and 3CLpro are excellent targets for the development of therapeutic antivirals, and have developed both the first non-covalent inhibitors of PLpro that show good antiviral activity, and inhibitors of 3CLpro with significant antiviral activity. We now propose to continue our research in a tightly focused proposal for the respective development of PLpro and 3CLpro inhibitors as effective SARS-COV therapeutics through the following overall specific aims: (1) Improve the current micromolar antiviral activity of our non-covalent PLpro inhibitors to low nanomolar levels through a process of structure-based molecular design and synthetic chemical improvement, and continue to identify alternate chemical scaffolds for PLpro inhibitors through computational screening and experimental validation as additional candidate leads in the event of toxicity or other biological concerns;(2) Improve the enzymatic inhibition and antiviral activity of our current non-covalent 3CLpro inhibitor leads through a process of structurebased molecular design and synthetic chemical improvement with a goal of low nanomolar antiviral activity, and continue to identify alternate chemical scaffolds for 3CLpro inhibitors through computational screening and experimental validation as backup candidates;(3) Identify mechanisms of resistance for both PLpro and 3CLpro inhibitors through viral passage to develop resistant mutants, identify the resulting mutations, and utilize structure-based design to reduce resistance potential;and (4) Evaluate candidate inhibitors for bioavailability and ADMET characteristics, and enhance the biological profile through chemical improvement; and evaluate efficacy of candidate compounds through animal challenge experiments, and develop further cycles of chemical improvement as needed. This proposed renewal builds on a well-established collaboration between Drs. Ghosh and Johnson, and incorporates a biology team that includes Dr. Wentworth for virology, Dr. Fung for enzymology and Dr. Weber for structural biology, and Dr. Jeong for ADMET. This team provides a tightly focused development strategy for new and effective SARS-COV therapeutics. The end goal of this application will be at least two lead inhibitors optimized to carry forward to extensive animal model evaluation for at least one to meet the two animal model approval criteria for SARS-CoV therapeutics. Additional evaluations against other coronavirus pathogens will provide data for potential broad spectrum application.